This invention was made with Government support under Grants Numbers CHE-8616907 and CHE-8620293 awarded by the National Science Foundation and under Grant Number GM-16609 awarded by the National Institutes of Health. The Government has certain rights in the invention.
The present invention relates, in general, to a method for enhancing the sensitivity of mass spectrometry, and more particularly, to a method of mass analysis of molecular samples utilizing Hadamard transform differences to obtain improved signal to noise ratios and increased accuracy and reliability in sample analysis.
The identification of compounds in complex mixtures and the structural elucidation of large molecules present difficult challenges in chemical analysis. Tandem mass spectrometry, or MS/MS, which involves the characterization of individual primary ions of a normal mass spectrum through their secondary product ions, greatly increases the information available from a sample, and is widely used for solving difficult problems of identifying trace components in complex mixtures. However, in conventional tandem mass spectrometry, the analysis of a sample material having many components is hampered by the great loss in sensitivity encountered when the spectrum of each precursor must be measured individually while discarding the precursor ions of all other precursor components. When analysis time or sample quantity is limited, the use of this technique may be limited to just a few precursors. MS.sup.n spectra in which mass-selected products are dissociated to produce further spectra, present an even greater measurement problem, since the number of possible dissociation or reaction pathways increases exponentially with n.
In conventional mass spectrometry, only a single primary ion from the sample is selected at one time for analysis, and any nonselected ions in the sample are lost. In order to analyze the sample completely, the mass selection of primary ions by conventional spectrometers is varied as a function of time so that (for example) higher and higher masses are selected for measurement of their secondary mass spectra. Over a period of time, all of the constituent ions are selected in sequence, and the entire primary spectrum of the target is covered, but this is time consuming and wasteful of energy and material.
When a Fourier transform technique is applied to a large number of components, the technique is hampered by a large loss of sensitivity, for when the selected precursors are measured individually, the precursor ions of other components are discarded. When the analysis time or the sample quantity is limited, such as is the case for on-line trace analysis for toxic substances in incinerator exhaust, peptide sequencing, etc., the use of this technique becomes limited to just a few precursors. In cases in which mass-selected products are then dissociated to produce even further spectra, an even greater measurement problem is encountered, since the number of possible dissociation or reaction pathways increases exponentially.
An improvement of this procedure involves the use of a two-dimensional Fourier transform mass spectrometry (FTMS), which can be used to measure simultaneously all of the parent and daughter ions which are selected, resulting in enhanced sensitivity for the collection of mass spectra. The multichannel detection capability of FTMS makes possible the collection of a complete secondary ion spectrum of a single parent ion, with nearly the same efficiency as the detection of a single one of the secondary ions. For example, as described by P. Pfandler et al in Chem. Phys. Lett. 1987, Vol. 138, pp. 195-200 and in Journal of Am. chem. Soc. 1988, Vol. 110, pp. 5625-5628, ions in an ion cyclotron resonance mass spectrometer are initially excited by RF energy to place precursor ions into cyclotron orbits wherein the frequencies of the orbits are functions of ion mass. A de-excitation RF pulse is applied after a specific delay time t.sub.1 to change the effective abundance of specific precursor ions as a function of the phase difference, which is dependent on the precursor mass to charge ratio (m/z value). Secondary mass spectra are then recorded as a function of t.sub.1 and the Fourier transform of this function for a specific mass peak then shows the abundance originating from each precursor. The major limitation of the Pfandler et al two-dimensional FTMS technique is that high resolution selection of parent ions which are present over a wide mass range requires very large numbers of MS-II spectra to provide sufficient resolution in determining which daughters come from their corresponding parent. This method has been demonstrated for larger ions, but never with parent ions separated by more than few Daltons, thereby seriously limiting any practical applications.
In accordance with the invention described in U.S. Ser. No. 07/241,869, molecular samples are measured through the use of a Hadamard transform wherein primary gaseous ions are produced from a molecular sample and different combinations of one half of those primary (or precursor) ions are selected. The selected primary ions are reacted, for example, by collisionally activated dissociation, to obtain from each of the primary ions a multiplicity of secondary, or daughter ions, of masses different than the masses of their precursor ions. The secondary ions are then separated in accordance with their masses and the abundances of the secondary ions are measured to obtain a mass spectrum of the secondary ions. This process is repeated to select different combinations of each primary ions, each time selecting one-half the total number of precursor ions, with the process being repeated the same number of times as there are primary ions. Upon completion of that process, the abundance of each secondary ion arising from the reaction of each primary ion is calculated by analyzing, through the use of the Hadamard transform, the yields of secondary ions from each combination of primary ions. Based on these yields, the constituents of the molecular sample can be identified with considerable accuracy and reliability. This reliability is enhanced by the fact that the repetitive measurements of different combinations of primary ions enhance the signal-to-noise ratio, allowing a gain of n.sup.0.5 /2 in measuring n primary ions, as compared to other techniques of analysis.
The invention described in Ser. No. 07/241,869, is an improvement over prior Fourier transform methods, for with the described Hadamard transform method only half of the precursor ions must be discarded in the course of a measurement, with the other half being selected, dissociated simultaneously, and the spectrum of the combined products measured. Thus, for example, if there are n primary ions, n/2 of these ions are selected. The selected primary ions are dissociated as by means of a collisionally activated dissociation reaction and the secondary ions are then mass-separated by means of a second stage of mass spectrometry. This process is then repeated for another combination of primary ions, again selecting a different n/2 ions and the mass yields are again obtained. This process is repeated a total of n times, each time selecting a different combination of primary ions. The abundance of a specific mass peak in a spectrum represents the sum of contributions from each precursor used; thus, each individual precursor contribution can be calculated using the corresponding abundance values from the n combined spectra, solving n simultaneous equations for n unknowns, with the process being repeated for each specific mass peak.
The Hadamard method described in Ser. No. 07/241,869 requires the use of instruments which have the ability to select and simultaneously dissociate any combination of precursor ions. This ability is an advantage of ion trap and Fourier-transform mass spectrometry which has received little attention in the past. FTMS also has the further advantage of multiple precursor selection at high resolution over a wide mass range using stored wave form inverse Fourier-transform (SWIFT) excitation as described by L. Chen et al in Analytical Chemistry 1987, 59, pp. 449-454 and by A.G. Marshall et al in Fourier Transform Mass Spectrometry; M.V. Buchanan, editor, American Chemical Society Symposium Series, No. 359; Washington, D.C. 1987; pp. 21-33. Such on/off selection of precursors for multichannel dissociation provides the information relating precursors to their corresponding daughters in each of the series of combined MS-II spectra for the Hadamard transform method. Precursor modulation for this purpose utilizes a sine function for the Fourier transform method. However, in both the Hadamard transform and the Fourier transform methods, the on/off or the sine function modulation of the precursors results in the loss of approximately one-half the detector signal.